Materials for hydrophilic interaction chromatography and processes for preparation and use thereof for analysis of glycoproteins and glycopeptides

Abstract

The invention relates to poly-amide bonded hydrophilic interaction chromatography (HILIC) stationary phases and novel HILIC methods for use in the characterization of large biological molecules modified with polar groups, known to those skilled in the art as glycans. The invention particularly provides novel, poly-amide bonded materials designed for efficient separation of large biomolecules, e.g. materials having a large percentage of larger pores (i.e. wide pores). Furthermore, the invention advantageously provides novel HILIC methods that can be used in combination with the stationary phase materials described herein to effectively separate protein and peptide glycoforms by eliminating previously unsolved problems, such as on-column aggregation of protein samples, low sensitivity of chromatographic detection of the glycan moieties, and low resolution of peaks due to restricted pore diffusion and long intra / inter-particle diffusion distances.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a Continuation-In-Part of U.S. patent application Ser. No. 14 / 576,682, filed Dec. 19, 2014, which application claims the benefit of priority of U.S. Provisional Patent Application No. 61 / 920,677, filed Dec. 24, 2013, the disclosure of each of which is expressly incorporated herein by reference thereto.BACKGROUND OF THE INVENTION[0002]Hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC) is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. The stationary phase of HILIC is a polar and hydrophilic phase which results in enhanced retention for polar analytes. The mobile phase of HILIC is a reversed-phase type high organic eluent, for example, a mixture of water and acetonitrile. A mechanism of separating analytes in HILIC can be a combination of partition...

AI Technical Summary

Benefits of technology

The invention provides a new type of material for use in chromatographic processes, specifically for the separation of large biomolecules such as glycans. These materials have high resolution, desirable retentivity, and selectivity of glycans. The invention also provides methods for using these materials to effectively separate protein and peptide glycoforms. The materials are poly-amide bonded, which provides advantages such as efficient separation of large biomolecules and non-retention of interfering analytes. The invention also includes a porous material with a copolymer comprising a hydrophilic monomer and a poly-amide bonded phase, which can be used in chromatographic processes. Overall, the invention provides new tools for the analysis of glycans and other biomolecules.

Problems solved by technology

Similarly, the authors of, “Separations of Intact Glycoproteins by HILIC” at the 33rd International Symposium and Exhibit on the Separation and Characterization of Biologically Important Molecules, Jul. 17-19, 2013 in Boston, Mass., USA, expressed that glycoprotein separations by HILIC would be of significant interest though at present this poses a significant challenge.

Their presented work was limited to separation of small (<20 kDa) glycoproteins.

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